Biomedical subjects

Laurie A Davidson

Publications and source records attributed to Laurie A Davidson.

12 recordsLinked to original sources

PubMed2005

A two-stage normalization method for partially degraded mRNA microarray data.

MOTIVATION: The goal of the study is to obtain genetic information from exfoliated colonocytes in the fecal stream rather than directly from mucosa cells within the colon. The latter is obtained through invasive procedures. The difficulties encountered by this procedure are that certain probe information may be compromised due to partially degraded mRNA. Proper normalization is essential to obtaining useful information from these fecal array data. RESULTS: We propose a new two-stage semiparametric normalization method motivated by the features observed in fecal microarray data. A location-scale transformation and a robust inclusion step were used to roughly align arrays within the same treatment. A non-parametric estimated non-linear transformation was then used to remove the potential intensity-based biases. We compared the performance of the new method in analyzing a fecal microarray dataset with those achieved by two existing normalization approaches: global median transformation and quantile normalization. The new method favorably compared with the global median and quantile normalization methods. AVAILABILITY: The R codes implementing the two-stage method may be obtained from the corresponding author.

Algorithms↗

PubMed2005

Folate transport gene inactivation in mice increases sensitivity to colon carcinogenesis.

Low dietary folate intake is associated with an increased risk for colon cancer; however, relevant genetic animal models are lacking. We therefore investigated the effect of targeted ablation of two folate transport genes, folate binding protein 1 (Folbp1) and reduced folate carrier 1 (RFC1), on folate homeostasis to elucidate the molecular mechanisms of folate action on colonocyte cell proliferation, gene expression, and colon carcinogenesis. Targeted deletion of Folbp1 (Folbp1(+/-) and Folbp1(-/-)) significantly reduced (P < 0.05) colonic Folbp1 mRNA, colonic mucosa, and plasma folate concentration. In contrast, subtle changes in folate homeostasis resulted from targeted deletion of RFC1 (RFC1(+/-)). These animals had reduced (P < 0.05) colonic RFC1 mRNA and exhibited a 2-fold reduction in the plasma S-adenosylmethionine/S-adenosylhomocysteine. Folbp1(+/-) and Folbp1(-/-) mice had larger crypts expressed as greater (P < 0.05) numbers of cells per crypt column relative to Folbp1(+/+) mice. Colonic cell proliferation was increased in RFC1(+/-) mice relative to RFC1(+/+) mice. Microarray analysis of colonic mucosa showed distinct changes in gene expression specific to Folbp1 or RFC1 ablation. The effect of folate transporter gene ablation on colon carcinogenesis was evaluated 8 and 38 weeks post-azoxymethane injection in wild-type and heterozygous mice. Relative to RFC1(+/+) mice, RFC1(+/-) mice developed increased (P < 0.05) numbers of aberrant crypt foci at 8 weeks. At 38 weeks, RFC1(+/-) mice developed local inflammatory lesions with or without epithelial dysplasia as well as adenocarcinomas, which were larger relative to RFC1(+/+) mice. In contrast, Folbp1(+/-) mice developed 4-fold (P < 0.05) more lesions relative to Folbp1(+/+) mice. In conclusion, Folbp1 and RFC1 genetically modified mice exhibit distinct changes in colonocyte phenotype and therefore have utility as models to examine the role of folate homeostasis in colon cancer development.

Animals↗

PubMed2005

Fish oil decreases oxidative DNA damage by enhancing apoptosis in rat colon.

To determine if dietary fish oil protects against colon cancer by decreasing oxidative DNA damage at the initiation stage of colon tumorigenesis, oxidative DNA damage, proliferation, and apoptosis were assessed by colonic crypt cell position using quantitative immunohistochemical analysis of 8-hydroxydeoxyguanosine (8-OHdG), Ki-67, and TUNEL assay, respectively. Sixty rats were provided one of two diets (corn oil or fish oil) and dextran sodium sulfate (DSS, an inducer of oxidative DNA damage) treatments (no DSS, 3% DSS, or DSS withdrawal). Fish oil feeding resulted in lower 8-OHdG levels (P = 0.038), higher levels of apoptosis (P = 0.035), and a lower cell proliferative index (P = 0.05) compared with corn oil feeding. In the top third of the crypt, fish oil caused an incremental stimulation of apoptosis with increased DNA damage (P = 0.043), whereas there was no such relationship with corn oil. Because polyps and tumors develop from DNA damage that leads to loss of growth and death control, the significant difference in fish oil vs. corn oil on these variables may account, in part, for the observed protective effect of fish oil against oxidatively induced colon cancer.

Animals↗

PubMed2004

Chemopreventive n-3 polyunsaturated fatty acids reprogram genetic signatures during colon cancer initiation and progression in the rat.

The mechanisms by which n-3 polyunsaturated fatty acids (PUFAs) decrease colon tumor formation have not been fully elucidated. Examination of genes up- or down-regulated at various stages of tumor development via the monitoring of gene expression relationships will help to determine the biological processes ultimately responsible for the protective effects of n-3 PUFA. Therefore, using a 3 x 2 x 2 factorial design, we used Codelink DNA microarrays containing approximately 9000 genes to help decipher the global changes in colonocyte gene expression profiles in carcinogen-injected Sprague Dawley rats. Animals were assigned to three dietary treatments differing only in the type of fat (corn oil/n-6 PUFA, fish oil/n-3 PUFA, or olive oil/n-9 monounsaturated fatty acid), two treatments (injection with the carcinogen azoxymethane or with saline), and two time points (12 hours and 10 weeks after first injection). Only the consumption of n-3 PUFA exerted a protective effect at the initiation (DNA adduct formation) and promotional (aberrant crypt foci) stages. Importantly, microarray analysis of colonocyte gene expression profiles discerned fundamental differences among animals treated with n-3 PUFA at both the 12 hours and 10-week time points. Thus, in addition to demonstrating that dietary fat composition alters the molecular portrait of gene expression profiles in the colonic epithelium at both the initiation and promotional stages of tumor development, these findings indicate that the chemopreventive effect of fish oil is due to the direct action of n-3 PUFA and not to a reduction in the content of n-6 PUFA.

Animals↗

PubMed2004

n-3 PUFA alter caveolae lipid composition and resident protein localization in mouse colon.

Caveolae, by virtue of their unique lipid environment, serve as signaling platforms that regulate cellular events. Perturbations in caveolae lipid composition have been shown in vitro to displace proteins from lipid microdomains, thereby altering their functionality and subsequent downstream signaling. Because membrane remodeling may not be accurately represented by using pharmacological treatments and in vitro models, we investigated the in vivo ability of dietary n-3 polyunsaturated fatty acids (PUFA) to alter caveolae lipid environment and the compartmentalization of resident proteins in mouse colonic mucosa. n-3 PUFA were examined for their chemoprotective, membrane lipid-modifying properties. Colonic caveolae in mice fed n-6 or n-3 PUFA enriched diets were characteristically enriched in cholesterol, sphingomyelin, and caveolin-1. n-3 PUFA feeding, compared with n-6 PUFA, significantly altered colonic caveolae microenvironment by increasing phospholipid n-3 fatty acyl content and reducing both cholesterol (by 46%) and caveolin-1 (by 53%), without altering total cellular levels. Concomitantly, localization of caveolae-resident signaling proteins H-Ras and eNOS in colonic caveolae was decreased by n-3 PUFA, by 45 and 56%, respectively. The distribution of non-caveolae proteins K-Ras and clathrin was unaffected. Moreover, EGF-stimulated H-Ras, but not K-Ras activation was significantly suppressed following n-3 PUFA feeding, in parallel with the selective alterations in their microlocalization. These findings reveal a novel modality by which n-3 PUFA remodel membrane microdomains in vivo and thereby alter caveolae protein localization and functionality.

Animals↗

PubMed2003

Dietary fish oil reduces oxidative DNA damage in rat colonocytes.

Prolonged generation of reactive oxygen species by inflammatory mediators can induce oxidative DNA damage (8-oxodG formation), potentially resulting in intestinal tumorigenesis. Fish oil (FO), compared to corn oil (CO), has been shown to downregulate inflammation and upregulate apoptosis targeted at damaged cells. We hypothesized FO could protect the intestine against 8-oxodG formation during dextran sodium sulfate- (DSS-) induced inflammation. We provided 60 rats with FO- or CO-supplemented diets for 2 weeks with or without 3% DSS in drinking water for 48 h. Half the treated rats received 48 additional h of untreated water before termination. Due to DSS treatment, the intestinal epithelium had higher levels of 8-oxodG (p =.04), induction of repair enzyme OGG1 mRNA (p =.02), and higher levels of apoptosis at the top of colonic crypts (p =.01) and in surface cells (p <.0001). FO-fed rats, compared to CO, had lower levels of 8-oxodG (p =.05) and increased apoptosis (p =.04) in the upper crypt region; however, FO had no significant effect on OGG1 mRNA. We conclude that FO protects intestinal cells against oxidative DNA damage in part via deletion mechanisms.

Animals↗

PubMed2003

Fish oil enhances targeted apoptosis during colon tumor initiation in part by downregulating Bcl-2.

We have shown that fish oil is protective against colon tumorigenesis, primarily by upregulating apoptosis. Production of prostaglandin E2 (PGE2) in colon cancer cells by cyclooxygenase (COX)-I and -II is known to inhibit apoptosis by induction of bcl-2. Because we have shown that fish oil downregulates PGE2 and COX-II, we hypothesized that this upregulation of apoptosis would be coincident with a downregulation of bcl-2. Bcl-2 was localized within the colonic crypt by quantitative immunohistochemistry (IHC), and scraped colonic mucosa was used for immunoblot analysis of bcl-2. The tissue used for bcl-2 analysis was from the rats used to determine apoptosis. Briefly, tissues were collected from rats consuming diets containing either corn oil or fish oil at 3, 6, 9, and 12 h after carcinogen injection. The correlation between bcl-2 and apoptosis was also determined. Bcl-2 expression decreased until 9 h (P < 0.05), whereas apoptosis increased until 9 h (P < 0.01). Bcl-2 expression and apoptosis were negatively correlated in both the proximal (P < 0.05) and distal colon (P < 0.005). Fish oil decreased bcl-2 expression (P < 0.05) and increased apoptosis (P < 0.05) in the top third of the crypt in the distal colon. In conclusion, one pathway by which fish oil may mediate apoptosis and thus protect against colon tumorigenesis is by downregulation of anti-apoptotic bcl-2.

Analysis of Variance↗

PubMed2002

Oncogenic ras alters sensitivity of mouse colonocytes to butyrate and fatty acid mediated growth arrest and apoptosis.

Docosahexaenoic acid (DHA) and butyrate favorably modulate colonocyte proliferation and apoptosis. In order to elucidate how oncogenic Ras modulates responses to these chemopreventive nutrients, we incubated isogenic non-transformed and Ras malignant transformed mouse colon cells with butyrate and DHA or linoleic acid (LA). Combining DHA with 1mM butyrate decreased proliferation relative to LA or no PUFA treatment in both cell lines. At a higher butyrate dose (5mM), caspase 3 activity was elevated to a greater extent in Ras transformed cells. Only non-transformed cells were sensitive to the apoptogenic effects of DHA, indicating that Ras transformation alters sensitivity to dietary chemopreventive agents.

Animals↗

PubMed2002

Protein kinase C betaII and TGFbetaRII in omega-3 fatty acid-mediated inhibition of colon carcinogenesis.

Increasing evidence demonstrates that protein kinase C betaII (PKCbetaII) promotes colon carcinogenesis. We previously reported that colonic PKCbetaII is induced during colon carcinogenesis in rodents and humans, and that elevated expression of PKCbetaII in the colon of transgenic mice enhances colon carcinogenesis. Here, we demonstrate that PKCbetaII represses transforming growth factor beta receptor type II (TGFbetaRII) expression and reduces sensitivity to TGF-beta-mediated growth inhibition in intestinal epithelial cells. Transgenic PKCbetaII mice exhibit hyperproliferation, enhanced colon carcinogenesis, and marked repression of TGFbetaRII expression. Chemopreventive dietary omega-3 fatty acids inhibit colonic PKCbetaII activity in vivo and block PKCbetaII-mediated hyperproliferation, enhanced carcinogenesis, and repression of TGFbetaRII expression in the colonic epithelium of transgenic PKCbetaII mice. These data indicate that dietary omega-3 fatty acids prevent colon cancer, at least in part, through inhibition of colonic PKCbetaII signaling and restoration of TGF-beta responsiveness.

Animals↗

PubMed2002

Dietary n-3 PUFA alter colonocyte mitochondrial membrane composition and function.

There is experimental evidence that dietary fish oil, which contains the n-3 fatty acid family, i.e., EPA and DHA, protects against colon tumor development, in part by increasing apoptosis. Since mitochondria can act as central executioners of apoptosis, we hypothesized that EPA and DHA incorporation into colonocyte mitochondrial membranes, owing to their high degree of unsaturation, would enhance susceptibility to damage by reactive oxygen species (ROS) generated via oxidative phosphorylation. This, in turn, would compromise mitochondrial function, thereby initiating apoptosis. To test this hypothesis, colonic crypts were isolated from rats fed either fish oil, purified n-3 fatty acid ethyl esters, or corn oil (control). Dietary lipid source had no effect on colonic mitochondrial phospholipid class mole percentages, although incorporation of EPA and DHA was associated with a reduction in n-6 fatty acids known to enhance colon tumor development, i.e., linoleic acid LNA) and its metabolic product, arachidonic acid (ARA). Select compositional changes in major phospholipid pools were correlated to alterations in mitochondrial function as assessed by confocal microscopy. The mol% sum of LNA plus ARA in cardiolipin was inversely correlated with ROS (P = 0.024). Ethanolamine glycerophospholipid ARA (P = 0.046) and choline glycerophospholipid LNA (P = 0.033) levels were positively correlated to mitochondrial membrane potential. In contrast, ethanolamine glycerophospholipid EPA (P = 0.042) and DHA (P = 0.024) levels were negatively correlated to mitochondrial membrane potential. Additionally, EPA and DHA levels in choline glycerophospholipids (P = 0.026) were positively correlated with caspase 3 activity. These data provide evidence in vivo indicating that dietary EPA and DHA induce compositional changes in colonic mitochondrial membrane phospholipids that facilitate apoptosis.

Animals↗

PubMed2002

Fish oil increases mitochondrial phospholipid unsaturation, upregulating reactive oxygen species and apoptosis in rat colonocytes.

We have shown that a combination of fish oil (high in n-3 fatty acids) with the butyrate-producing fiber pectin, upregulates apoptosis in colon cells exposed to the carcinogen azoxymethane, protecting against colon tumor development. We now hypothesize that n-3 fatty acids prime the colonocytes such that butyrate can initiate apoptosis. To test this, 30 Sprague-Dawley rats were provided with diets differing in the fatty acid composition (corn oil, fish oil or a purified fatty acid ethyl ester diet). Intact colon crypts were exposed ex vivo to butyrate, and analyzed for reactive oxygen species (ROS), mitochondrial membrane potential (MMP), translocation of cytochrome C to the cytosol, and caspase-3 activity (early events in apoptosis). The fatty acid composition of the three major mitochondrial phospholipids was also determined, and an unsaturation index calculated. The unsaturation index in cardiolipin was correlated with ROS levels (R = 0.99; P = 0.02). When colon crypts from fish oil and FAEE-fed rats were exposed to butyrate, MMP decreased (P = 0.041); and translocation of cytochrome C to the cytosol (P = 0.037) and caspase-3 activation increased (P = 0.032). The data suggest that fish oil may prime the colonocytes for butyrate-induced apoptosis by enhancing the unsaturation of mitochondrial phospholipids, especially cardiolipin, resulting in an increase in ROS and initiating apoptotic cascade.

Animals↗

PubMedDate not supplied

Quantification of human intestinal gene expression profiles using exfoliated colonocytes: a pilot study.

Early detection of colon cancer can result in a high cure rate; therefore, an accurate screening method is imperative. Adoption of non-invasive testing designed to reduce anxiety over colorectal cancer screening and improve early detection is highly desirable. Therefore, we have developed a novel non-invasive methodology utilizing exfoliated colonocytes in order to quantify colonic messenger RNAs (mRNAs). Previously we have demonstrated in the rat that intact eukaryotic mRNA can be isolated due to the presence of exfoliated colonocytes in the faecal stream. To assess use of this methodology in humans, this pilot study evaluated exfoliated colonocyte mRNA expression of 11 putative biomarkers using real-time reverse transcription-polymerase chain reaction (RT-PCR) in seven normal subjects, four subjects with inflammation, and 10 tumour-bearing subjects presenting for colonoscopy. Expression of the biomarkers was evaluated following normalization to TATA box binding protein mRNA levels. Tumour-bearing subjects diagnosed with adenoma had elevated levels of cyclin Dl (p = 0.041). In addition, subjects displaying inflammation of the colon exhibited higher mRNA levels of cyclooxygenase-2 (p = 0.007). These data suggest that mRNA isolated from exfoliated colonocytes could be used to detect early stages of colon cancer, and possibly chronic inflammation. To broaden the utility of non-invasive marker analysis, additional studies are needed to generate a multi-target assay panel of diagnostic markers. This will allow for the development of robust classifiers that can determine critical gene sets for the diagnosis and prediction of colon cancer in animal models and humans.

Adenoma↗